This revised proposal is a renewal application of our 25 year program that has contributed paradigm- shifting concepts for understanding molecular mechanisms regulating osteoblast commitment, growth and differentiation. We have established the stages of osteoblast differentiation and temporal control of the osteoblast phenotype by genetic and epigenetic mechanisms in vitro and using in vivo animal models. The contributions of this program include establishing bone tissue-specific properties of transcription factors, chromatin remodeling and histone modifications related to control of osteoblast-expressed mRNAs and noncoding RNAs. Our current program discovered that short noncoding microRNAs are key regulators of signaling pathways essential for bone formation. Little is known, however, about the identity and function of long noncoding (lncRNAs) involved in osteogenesis. The new direction of this renewal is to identify and characterize lncRNAs during osteoblastogenesis, and discover their molecular mechanisms and functional relevance in osteogenesis. lncRNAs have recently emerged as a novel level of gene regulation in development and lineage commitment. They function through complex mechanisms including acting as: (i) guides to recruit chromatin modifying enzymes to activate or repress transcription; (ii) scaffolds bringing proteins into close proximity to form activation or repression complexes; (iii) decoys to sequester transcription or other regulatory factors such as microRNAs; and (iv) enhancers to increase expression of neighboring genes. lncRNAs have been demonstrated to play roles in numerous biological contexts including adipogenesis, hematopoiesis and myogenesis. Likewise, we hypothesize that lncRNAs are essential for osteogenesis and regulate key developmental stages necessary for normal bone formation. Our preliminary results using RNA-Seq analysis have defined the mouse and human lncRNA transcriptomes during differentiation of mesenchymal stem cell (MSCs) to osteoblasts. We have identified conserved expression of human-mouse lncRNAs and selected a subset for mechanistic and functional characterization in vitro and in vivo. Preliminary analysis of lncRNA-knockout mice has revealed bone phenotypes. Our proposed aims will provide transformative insight into how lncRNAs contribute to the complex machinery regulating osteogenesis. Aim 1 will determine the function of human-mouse homologous lncRNAs in osteoblasts in vitro; Aim 2 will discover mechanisms by characterizing lncRNA interactomes; and Aim 3 will address the in vivo activity of select lncRNAs in mouse models. This innovative investigation of osteoblast lncRNAs will advance understanding of epigenetic control for bone formation. Our findings will impact the development of new dimensions for investigations that are relevant to human skeletal disease.